Synergism of amlodipine and telmisartan or candesartan on blood pressure reduction by using SynergyFinder 3.0 and probability sum test in vivo

Abstract This study was designed to evaluate the synergism of two couples of antihypertensive drugs (amlodipine + telmisartan and amlodipine + candesartan) on blood pressure reduction in vivo by both SynergyFinder 3.0 and probability sum test. Spontaneously hypertensive rats were treated with intragastric administration of amlodipine (0.5, 1, 2, and 4 mg/kg), telmisartan (4, 8, and 16 mg/kg), candesartan (1, 2, and 4 mg/kg), nine combinations for amlodipine and telmisartan, and nine combinations for amlodipine and candesartan. The control rats were treated by 0.5% carboxymethylcellulose sodium. Blood pressure was recorded continuously up to 6 h after administration. Both SynergyFinder 3.0 and the probability sum test were used to evaluate the synergistic action. The synergisms calculated by SynergyFinder 3.0 are consistent with the probability sum test both in two different combinations. There is an obviously synergistic interaction between amlodipine and telmisartan or candesartan. The combinations of amlodipine and telmisartan (2 + 4 and 1 + 4 mg/kg) and amlodipine and candesartan (0.5 + 4 and 2 + 1 mg/kg) might exert an optimum synergism against hypertension. Compared with the probability sum test, SynergyFinder 3.0 is more stable and reliable to analyze the synergism.


| INTRODUC TI ON
Drug combinations have become a standard therapy for many complex diseases, including cancers and cardiovascular diseases. 1,2 The combination with different mechanism drugs might produce synergistic interaction. It can enhance the treatment efficacy and avoid the acquisition of monotherapy resistance. Good combination therapy also reduces the dose of each drug. It is helpful to minimize the clinical and metabolic side effects of each individual component in larger dosage. 3 SynergyFinder, a web-application, was put forward in 2017 and has been widely used for the discovery of novel synergistic drug combinations. 4,5 SynergyFinder was designed to calculate the synergism between anti-cancer medicines first. It also can be used in many precision medicine areas, targeted drug combination discovery. 6 Recently, we reported that it can be used for anti-platelet aggregation in vitro. 7 It is unclear whether this method can be used to evaluate the synergism of antihypertensive drugs in vivo.
For the combination of different antihypertensive drugs, the probability sum test (q test) has been suggested to evaluate the synergism. 8,9 This test derives from classic probability analysis, and several groups have suggested that it may be useful for evaluating the synergism of combinations of two drugs in vivo. [8][9][10][11][12][13] However, there are some disadvantages.
For example, about 20% data are discarded during the calculation. This elimination might affect the accuracy of the synergistic conclusion.
In the present study, both SynergyFinder 3.0 (the latest upgrade) and the probability sum test were performed to analyze the synergism on blood pressure reduction in vivo. Two couples of antihypertensive drugs (amlodipine + telmisartan and amlodipine + candesartan) were used in spontaneously hypertensive rats (SHRs). We mainly focused in the difference between the probability sum test and SynergyFinder 3.0 for the evaluation of synergism on blood pressure reduction.

| Animals
Male SHRs (aged 3 months) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed with controlled temperature (23-25°C) and lighting (8:00 AM to 8:00 PM light, 8:00 PM to 8:00 AM dark) and with free access to food and tap water. All the animals used in this experiment received humanitarian concern. All animal experiments were approved by the Committee on ethics of biomedicine of Naval Medical University and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

| Blood pressure continuous recording in conscious rats
The operation and measurement were conducted as described previously in detail. 2,14 Briefly, rats were anesthetized by inhalation of 5% isoflurane and maintained with 2% isoflurane.
The lower abdominal aorta was catheterized via the left femoral artery with a polyethylene catheter full of heparin (100 U/ mL) for the recording of blood pressure. The catheters were tunneled subcutaneously, exposed through the interscapular skin and fixed. Then the animals were treated by meloxicam (2 mg/ kg, subcutaneous injection) and penicillin (80 000 IU per animal, intramuscular injection) to relieve pain and prevent infection for 2 days. After 2 days recovery (with free access to food and tap water), rats were placed individually in cylindrical cages for blood pressure recording. The aortic catheter was connected to a pressure transducer via a swivel allowing rats to move freely. Beat-tobeat blood pressure signals and heart period were digitized by the MPA-HBBS system (Alcott Biotech Co. Ltd) mounted in computer.
After approximately 3 h habituation, the blood pressure and heart period signals were determined online. The data of blood pressure from 12:00 to 13:00 are used as the baseline (before administration). Then, at 13:00, the drug was given via the catheter of gastric fistula. The time reaching the maximum reduction in the blood pressure of each group was about half hour. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) values from every heart beat were recorded from 0.5 h after administration up to 6 h. The drugs were dissolved in the 0.5% carboxymethylcellulose sodium (CMC) and were given into the stomach through a catheter. The blood pressure was recorded according aforementioned method. The blood pressure values an hour before administration and mean values 6 h after administration were recorded to analyze. Male SHRs were divided into 13 groups (n = 10 per group) and administered by the following drugs: amlodipine (0.5 mg/kg), candesartan (1, 2 and 4 mg/kg) and the combinations (0.5 + 1, 0.5 + 2, 0.5 + 4, 1 + 1, 1 + 2, 1 + 4, 2 + 1, 2 + 2 and 2 + 4 mg/kg).
The drugs were dissolved in the 0.5% CMC and were given into the stomach through a catheter. The blood pressure was recorded according aforementioned method. Blood pressure data of other 2 amlodipine alone groups (1 and 2 mg/kg) were got from experiment 1.
Another 10 SHRs were treated by vehicle (0.5% CMC) with the same volume as a control group. The blood pressure was recorded accordingly. The blood pressure values 1 h before administration and mean values 6 h after administration were recorded to analyze.

| SynergyFinder 3.0
An excel was set up. There are several lines, including "Pair Index", "Drug 1", "Drug 2", "dose 1", "dose 2", "Response" and "Dose Unit". "Pair Index" represents how many pairs of drugs. "Drug 1" and "Drug 2" are the name of drugs in a dose-response, while "dose 1" and "dose 2" are the doses of the drugs in combination. "Response" means the effect of drug combinations at the doses input by "dose 1" or "dose 2". It is the reduction percentage of blood pressure. The range of the "Response" is expected from 0 to 100. The "Dose Unit" means the unit of drugs' dose.
The inhibition ratio of blood pressure reduction was used for SynergyFinder 3.0 system. Inhibition ratio = (blood pressure after administration − blood pressure before administration) × 3.33/blood pressure before administration × 100%. Unlike tumor cells, blood pressure cannot be reduced or inhibited by 100%. About 30% reduction in blood pressure is most appropriate in clinical. 15 So we defined a 30% reduction in blood pressure as 100% inhibition. Dose-effect curves for each drug on blood pressure were drawn and the half maximal inhibitory dose were calculated by using Prism software, version 8 (GraphPad). Choosing the percentage reduction as the phenotypic response, the blood pressure reduction of each drug combination was analyzed using SynergyFinder 3.0 (https://syner gyfin der.fimm.fi). 5 Briefly, the mean percentage of blood pressure reduction of a pair of antihypertensive drugs, and their combinations at different doses were input respectively. According to the software recommendation, highest single agent (HSA) reference model was selected in this study. This model states the expected combination effect is the maximum of the single drug responses at corresponding concentrations. Finally, the comprehensive synergy score and synergy maps of drug combination were obtained automatically by the software using HSA model. The synergy score was used to evaluate the efficacy of the combination. According to the user guide in the website, synergy score was evaluated as follows: scores <−10: the interaction between two drugs was likely to be antagonistic; from −10 to 10: the interaction between two drugs was likely to be additive; and > 10: the interaction between two drugs was likely to be synergistic. 7 The synergy scoring can be visualized as either a two-dimensional or a three-dimensional interaction surface over the dose matrix. The depth of the color reveals the degree of percentage of blood pressure reduction in the two-dimensional image and the height of the 3D drug interaction landscape is standardizing as the percentage of blood pressure reduction to show the comparison of the degrees of interaction among drug combinations.

| Probability sum test
The probability sum test was used to evaluate the synergism of the combination according to the references. [10][11][12] Compared with the control group, the experimental groups with a decrease in blood pressure >15 mmHg were defined as responders. Similarly, rats with a decrease in blood pressure <12 mmHg (80% of 15 mmHg) were defined as non-responders and blood pressure data ranging from 12 to 15 mmHg were discarded. We calculate the q value as follows: q = P A+B /(P A + P B − P A × P B ). A and B represent drug A and drug B. P is the percentage of responders in each group. P A+B is the responders' real percentage and (P A + P B − P A × P B ) is the expected response ratio. (P A + P B ) is the sum of the probabilities when drug A and drug B are used alone. P A × P B is the probability of ratios responding to both drugs when they were used alone. We considered that the combination was synergistic when q was >1.15 and the combination was antagonistic when q was <0.85. When q was between 0.85 and 1.15, the combination was additive.

| Statistical analysis
The investigators were blinded to the animal groups when they assessed the blood pressure and analyzed the synergistic score using SynergyFinder 3.0 or the probability sum test. The animals were randomly assigned by using the random permutations table.
Continuous variables were expressed as mean ± standard deviation (SD). Student's t-test (two-tailed paired t-test) was used for comparisons between before and after drug administration. p < .05 was considered statistically significant.

| Effects of amlodipine, telmisartan alone, and their combination on SBP, DBP, and MBP in SHRs
The effects of a single dose of amlodipine and telmisartan alone or in their combinations on SBP, DBP, and MBP are shown in Figure 1. In amlodipine alone treatment groups, compared to before administra-       Table 1 Table 2 shows the probability sum test of SBP, DBP, and MBP in data

| DISCUSS ION
Amlodipine is a calcium channel blocker (CCB). 16 Telmisartan and candesartan belong to angiotensin II type 1 (AT (1)) receptor blocker (ARB). 17 The action mechanisms between amlodipine and telmisartan or candesartan are also quite different. They might have synergistic effect on blood pressure reduction.   The probability sum test also has been widely used to analyze the synergism of the combinations of two antihypertensive drugs. [8][9][10][11][12][13] The synergistic or antagonistic effect is mainly defined by the q value (P A+B /(P A + P B − P A × P B )). However, there are some disadvantages for this method compared with SynergyFinder 3.0.
First, the definition of responders for blood pressure reduction.
We usually define a decrease in blood pressure >15 or 20 mmHg as responders. 2,10 P is the percentage of responders in each group. When the reduction percentage is equal to zero, it will have a great impact on the q values, which might cause a big difference between different responders. Second, the elimination TA B L E 2 The probability sum test in rats administrated with amlodipine and candesartan or their combinations. of some data might affect the accuracy of q values. In this experiment, a reduction in blood pressure >15 mmHg is defined as responders. A decrease <12 mmHg (80% of 15 mmHg) is defined as non-responders and data ranging from 12 to 15 mmHg are discarded, which might affect the synergistic conclusion.
In SynergyFinder 3.0, all the data are input to the system and are reflected in synergy pictures and scores. SynergyFinder 3.0 combines multiple synergy scoring models and obtains a novel consensus synergy score. During the data processing, the false positive results will be removed with an improved outlier detection functionality. 5 Hence, individual extreme data will not have much influences on synergy scores. Different with the probability sum test, the synergy scores of SBP, DBP, and MBP by SynergyFinder 3.0 are high consistent.
In conclusion, SynergyFinder 3.0 is more stable and reliable to analyze the synergism on blood pressure reduction in vivo than the probability sum test. There is an obviously synergistic interac-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
Available per request.

E TH I C S S TATEM ENT
All experiments were approved by the Committee on ethics of biomedicine of Naval Medical University and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.